Journal: Frontiers in Molecular Biosciences

Article Title: Increased Potency of a Bi-specific TL1A-ADAM17 (TACE) Inhibitor by Cell Surface Targeting

doi: 10.3389/fmolb.2017.00061

Figure Lengend Snippet: The A2 bi-specific inhibitor exhibits similar biochemical activity as the two mono-specific H3 (DR3) and pTACE inhibitors. (A) Binding of A2 to TL1A in comparison to the mono-specific H3 protein. Binding analysis was performed by ELISA using plates that were pre-coated with TL1A (see Section Materials and Methods for detailed description). (B) Inhibition of TACE by A2 in comparison to the mono-specific pTACE inhibitor. Inhibition of TACE activity was assessed using a fluorogenic peptide substrate DEVD-AMC.

Article Snippet: The macrophages medium was collected and the TNF-α in the medium was determined using mouse TNF-α DueSet ELISA kit (R&D systems) as recommended by the manufacturer.

Techniques: Activity Assay, Binding Assay, Comparison, Protein Binding, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: Frontiers in Molecular Biosciences

Article Title: Increased Potency of a Bi-specific TL1A-ADAM17 (TACE) Inhibitor by Cell Surface Targeting

doi: 10.3389/fmolb.2017.00061

Figure Lengend Snippet: Similar inhibition level of TNF-α release from macrophages following incubation with A2 and the mono-specific pTACE inhibitor. The levels of TNF-α secretion from mouse peritoneal macrophages were determined following 3 h of LPS stimulation at a concentration of 2.5 ng/ml with or without 1 μM of A2 or pTACE. Media supernatant was analyzed by ELISA for detection of TNF-α levels, the TNF-α values were calculated according to TNF-α calibration curve, * P < 0.05.

Article Snippet: The macrophages medium was collected and the TNF-α in the medium was determined using mouse TNF-α DueSet ELISA kit (R&D systems) as recommended by the manufacturer.

Techniques: Inhibition, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Increased Potency of a Bi-specific TL1A-ADAM17 (TACE) Inhibitor by Cell Surface Targeting

doi: 10.3389/fmolb.2017.00061

Figure Lengend Snippet: Increased potency of A2 vs. H3 (DR3) in inhibiting TL1A induced IFN-γ secretion and apoptosis in PBL and TF-1 cell line, respectively. (A) Inhibition of TL1A-induced secretion of IFN-γ in human PBL by increased concentration of A2 and H3. Cells were incubated for 72 h with 200 ng/ml TL1A, 20 ng/ml IL-12 and 50 ng/ml IL-18, and different concentrations of A2 and H3 inhibitors. The 1:10 diluted cell supernatant was analyzed by ELISA for detection of IFN-γ levels. The IFN-γ values were calculated according to IFN-γ calibration curve. (B) Inhibition of TL1A-induced apoptosis in TF-1 cells by increased concentration of A2 and H3. Cells were incubated for 6 h with 8 μg/ml of cyclohexamide (CHX) and 75 ng/ml of TL1A and the indicated concentration of A2 and H3 receptors. Following incubation, lysis buffer containing the caspase-3 fluorescent substrate DEVD-AMC was added and enzyme activity was monitored for 10 min. The data presented in the PBL and TF-1 experiments is the average of three independent repeats of each experiment and the error bars represent the standard deviation from the average.

Article Snippet: The macrophages medium was collected and the TNF-α in the medium was determined using mouse TNF-α DueSet ELISA kit (R&D systems) as recommended by the manufacturer.

Techniques: Inhibition, Concentration Assay, Incubation, Enzyme-linked Immunosorbent Assay, Lysis, Activity Assay, Standard Deviation